Antimicrobial activity of cell free supernatants from probiotics inhibits against pathogenic bacteria isolated from fresh boar semen

The use of antibiotics with semen extender appears to be a practical solution to minimise bacterial growth in fresh boar semen preservation. Unfortunately, the excessive use of antibiotics promotes antimicrobial resistance (AMR). This becomes a worldwide concern due to the antimicrobial resistance genes transmitted to animals, environment, and humans. Probiotics are one of the alternative methods to reduce antibiotic use. They could inhibit pathogenic bacteria by producing antimicrobial substances in cell free supernatants (CFS). Nevertheless, there is no comprehensive study undertaken on inhibitory activity against pathogenic bacteria isolated from boar semen origin. Our study investigated the efficacy of CFS produced from selected probiotics: Bacillus spp., Enterococcus spp., Weissella spp., Lactobacillus spp., and Pediococcus spp. inhibiting pathogenic bacteria isolated from fresh boar semen. Besides, the semen-origin pathogenic bacteria are subjected to identification, antimicrobial resistance genes detection, and antibiotic susceptibility test (AST). Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis are the most common pathogens identified in boar semen with resistance to numerous antibiotics used in pig industry. The CFS with its antimicrobial peptides and/or bacteriocin constituent derived from selected probiotics could inhibit the growth of pathogenic bacteria carrying antimicrobial resistance genes (mcr-3 and int1 genes). The inhibition zones for Pseudomonas aeruginosa, Escherichia coli, and Proteus mirabilis provided more efficient results in the CFS derived from Lactobacillus spp. and Pediococcus spp. than those of the CFS produced from Enterococcus spp., Weissella spp. and Bacillus spp., respectively. It is worth noted that as the incubation time increased, the antibacterial activity decreased conversely. Our results on CFS with its antimicrobial peptides and/or bacteriocin constituent inhibits semen-origin pathogenic bacteria guide the direction as a promising alternative method used in the semen extender preservation of the pig industry.

Detection of mcr and int1 genes. The detection of mcr-1 to mcr-10 and int1 in all pathogens isolated from fresh boar semen were performed using multiplex PCR and showed in Table 1. The int 1 gene positive was detected in 3 strains of P. mirabilis, 1 isolate of each P. aeruginosa, E. coli, C. koseri and E. hormaechei from both Farm A and B. Most pathogens in this study were not contained mcr-1 to mcr-10 genes except only one E. coli isolate from Farm B showed positive to mcr-3. This E. coli strain also exhibited the int 1 positive and was classified as multidrug resistant according to MIC result. All pathogens which are carried either int 1 or mcr-3 were classified as multidrug resistant strains based on the MIC result as shown in Table 1.
Cell free supernatants (CFS) against pathogens from boar semen. The three selected pathogens from boar semen (P. aeruginosa, E. coli, and P. mirabilis) were conducted to find out the inhibitory effect of CFS from probiotics. The CFS was collected from eight interesting probiotics and their characteristics were declared in Table 2. The antimicrobial activities of CFS were determined as inhibition zone by performing agar well diffusion assay (Fig. 1).
The inhibitory activity of CFS against P. aeruginosa could be initially noticed at 8 h incubation except for the CFS from B. subtilis (Fig. 2a). At 10 h of incubation, there were no longer inhibitory effects of CFS from B. axarquiensis and B. amyloliquefaciens. On the contrary, the CFS produced from E. faecium, L. plantarum, P. acidilactici, P. pentosaceus, and W. confusa could remain the inhibitory effect with the presence of similar The interesting probiotics could produce CFS with inhibitory effects against E. coli except for CFS derived from B. axarquiensis (Fig. 2b). In details, the CFS produced from B. amyloliquefaciens and B. subtilis could inhibit E. coli in short period of time, 8-10 h and 8 h respectively. On the contrary, the CFS produced from E. faecium, L. plantarum, P. pentosaceus, and W. confusa thoroughly expressed the inhibitory effects from 8-16 h incubation time. Interestingly, it was apparent that CFS from P. acidilactici exhibited the largest inhibition zone (27.00 to 32.00 mm) against E. coli. Furthermore, the inhibitory effects of CFS produced from probiotics against E. coli shared the same tendency to P. aeruginosa.
Finally, the CFS produced by B. axarquiensis and B. subtilis could not show the inhibitory activities against P. mirabilis at 8-16 h incubation time (Fig. 2c). At 8-10 h incubation time, the diameter of inhibition zone expressed from L. plantarum, P. acidilactici, P. pentosaceus, and W. confusa were statistic equivalently. Interestingly, the CFS   www.nature.com/scientificreports/ produced by P. acidilactici still exhibited the largest diameter of inhibition zone (27.00 to 28.00 mm) thorough the incubation period. In contrary, the CFS from B. amyloliquefaciens were presented inhibition activity with a smallest inhibition zone (13.00 to 17.00 mm). Ultimately, it was apparent that the inhibitory activities of probiotics derived CFS shared the same tendency against P. aeruginosa, E. coli, and P. mirabilis. In other words, the inhibitory activities became less effective from the decline of inhibition zone when it had continued for a long period of incubation time.

Discussion
It was apparent that the bacteria in fresh boar semen could be identified as both Gram-negative and Grampositive bacteria. P. aeruginosa, E. coli, and P. mirabilis are the predominant bacteria in this study. The results of this survey are found to be similar to a previous survey undertaken in Brazil 38 .In addition, P. aeruginosa and E. coli caused negative effects on boar spermatozoa, whether to induce sperm agglutination or decrease sperm motility 10,38 . That effect could be limited by using gentamicin antibiotics in semen extender 10 . The antimicrobial susceptibility test revealed that P. aeruginosa and E. coli show susceptibility rates to gentamicin 25% and 50%, respectively, while P. mirabilis was susceptible to gentamicin for 100%. Additionally, it was observed that sows with endometritis had higher rates of the antimicrobial resistant bacteria. These findings should be of concern since endometritis can be transmitted by bacterial contamination in boar semen 1,37 . In their study, Burch and Sperling 40 found that 41% of endometritis sows were caused by a single bacterial infection, 72.3% of which were identified as E. coli. From the result, E. coli was the most resistant to common antibiotics including amoxicillin and tetracycline, which are used in pig farms and boar semen extender. The antimicrobial susceptibility test from fresh boar semen revealed that the majority of Gram-negative bacteria were resistant to antibiotic, whereas the Gram-positive bacteria were less antimicrobial resistance. The antibiotic resistance ratio from fresh boar semen was similar to a study undertaken in Italy 41 , while a study in Romania found 56.52% of Gram-negative boar semen bacterial isolation were resistant to gentamicin 42 . This study discovered a high rate of antibiotic drug resistance which increases concerns about the problems caused by the use of antibiotics in agriculture, while the use of antibiotics with semen extender is recommended to protect the spermatozoa 14 . The discovery of novel antimicrobial compounds such as antimicrobial peptides and/or bacteriocin derived from CSF to replace conventional antibiotics is an interesting issue to conduct in the future. It has been shown that the alternative methods were studied to find a feasible way to reduce the use of antibiotics in boar semen extender, including: (i) bacterial removal by a physical method using single-layer centrifugation 43 ; (ii) antimicrobial peptides (AMPs) or short antimicrobial lipopeptides 44 ; (iii) other substances, such as lysozyme and kojic acid 45,46 ; and (iv) semen storage at low temperature conditions (5 °C) without antibiotic supplement 47 . These methods have advantages and disadvantages, such as loss of spermatozoa from the physical method 43 or no broad-spectrum activity with kojic acid 46 . Moreover, each method has strengths and weaknesses in terms of antimicrobial activity and the effect on semen quality.
Although studies on bacterial contamination and antibiotic resistant bacteria from boar semen are available, no report of antimicrobial resistance genes, particular mcr-3 gene was found. One boar semen sample was detected for mcr-3. By considering the relationship between mcr-3 detection and colistin resistance, it was found that the sample was susceptible to colistin. The emergence of this incident resembles the study of mcr-1, mcr-4, and mcr-5 presented by García et al. 48 as well as mcr-1 to mcr-10 by Nguyet et al. 49 . These studies utilised five samples of mcr genes positive (one sample from mcr-1 and four samples from mcr-4) which were found to be susceptible to colistin 48 . The positive mcr gene without colistin resistant spectacle was possible because bacteria were a carrier of an inactive form of the mcr gene 48 . Meanwhile the result of four negative mcr genes were found to be colistin resistant by the MIC test (three samples from P. mirabilis and one sample from P. stuartii). The colistin resistant with the negative mcr gene corresponded to a previous study of E. coli 49 . The colistin resistance was supposed to have another mechanism that does not depend on mcr genes. Consequently, the higher incidence of mcr genes in livestock animals significantly increased the risk of mcr genes being transmitted to humans. In detail, the mcr genes could be transmitted to humans via foodborne, zoonotic, and vector-borne routes. Nevertheless, the incidence of the mcr gene in humans is higher than the incidence in animals 50 .
The prevalence of the int1 gene has been studied at different stages of the pig production system. The results indicate that there is a high-rate detection of the int1 gene in sows and piglets by conducting a rectal swab. Nevertheless, detection of the gene in boars has not been studied 51 . The present study successfully detected the int1 gene from boars. In addition, the int1 gene was detected from P. mirabilis, P. aeruginosa, E. coli, C. koseri, and E. hormaechei. The int1 gene has been detected in Gram-negative bacteria and resulted in various antimicrobial resistances, such as β-lactam, sulfonamide, and aminoglycoside 28,52 . According to the related literature, the int1 gene was not only detected from Gram-negative bacteria but also from Gram-positive bacteria including Staphylococcus spp. 53,54 . Furthermore, Stalder et al. 55 found that the presence of the int1 gene increased the risk of the spread and transmission of resistance genes to environment and other bacteria.
Our findings, one E. coli isolate was detected both of the mcr-3 and int1 genes. This result corresponded with a previous study 49 which had 24 out of 37 samples positive for both int1 and mcr genes, and four samples were only positive for the int1 gene.
The results of the antimicrobial effect of CFS from selected probiotics exhibited a decreased diameter of the inhibition zone as the incubation time continued. To support this finding, our results are consistent with previous studies 32,34,56 . In addition, Kaewchomphunuch et al. 32 reported the CFS from L. acidophilus, L. plantarum, and P. pentosaceus which expressed inhibitory activity only to pathogenic E. coli isolated from pigs. Nevertheless, the previous study did not undertake a comprehensive study of the inhibitory activity against other pathogens or specific E. coli strain isolated from semen origin.
The CFS produced from L. acidophilus could inhibit the growth of P. aeruginosa while another activity was able to disrupt biofilms from P. aeruginosa 56 . The difference between El-Mokhtar's study and the present study are the probiotics for CFS collection. Our study collected from other probiotics in lactic acid bacteria (LAB) including P. acidilactici, P. pentosaceus, and L. plantarum which showed similar results.
From the results of the present study, P. mirabilis could be inhibited by CFS from selected probiotics except for B. axarquiensis and B. subtilis. The inhibitory results are similar to Shaaban et al. 57 study which mentioned the action from L. casei and L. reuteri. Besides, the inhibitory activities of pathogen growth inhibition derived from the CFS of L. casei and L. reuteri were able to inhibit P. mirabilis biofilm formation as well.
Factors that affect the ability to inhibit pathogens that depend on pH or concentration of antimicrobial compound including lactic and acetic acid or antimicrobial peptides (AMPs) 57,58 . The comparable findings of CFS from L. johnsonii was between pH 3.5 and pH 6.0. In addition, it has been reported that lower pH (acidic condition) could inhibit growth of B. cereus while higher pH causes the activity to disappear 58 . The reduced antimicrobial activity of CFS from Lactobacillus spp. and Enterococcus spp. cultures were found when their pH value was greater than 4.5 59 . However, a study of CFS from L. plantarum adjusted the pH up to 6.5 and the antimicrobial activity was still maintained 34 . In the same direction, Soria and Audisio 58 revealed that the different compound in CFS also influenced the inhibitory activity of bacterial growth. From the present results, the pH value was observed as a main feature in the antimicrobial activity of CFS, the key compound in CFS for inhibiting pathogen growth might be that of AMPs and/or bacteriocins. With regard to the pH influence on inhibitory activity, our 3 CFSs produced from Lactobacillus plantarum NN31-5B, Pediococcus acidilactici NN82-7M and Pediococcus pentosaceus NN115-6M were neutralized to pH 6.0 and performed the antimicrobial activities with boar semen pathogens by agar well diffusion assay (data not shown). The inhibition zone was decreased but still inhibit the pathogens which showed similar results to other studies 34 www.nature.com/scientificreports/ might be contained antimicrobial peptides and/or bacteriocin with their antimicrobial properties. The AMPs were isolated from CFS (subtilosin) derived from B. amyloliquefaciens and could inhibit the bacterial vaginosis associated bacteria similar with the subtilosin from B. subtilis 60 . In addition, organic acid was found to be an essential compound of CFS derived from LAB. Nevertheless, Arrioja-Bretón et al. 34 mentioned that the acid was not the only factor inhibiting bacterial growth. To support this hypothesis, Tenea 61 demonstrated that the AMPs extracted from LAB-producing CFS (L. plantarum and Lactococcus lactis) could also inhibit against Salmonella enterica.
Although the previous studies were conducted to investigate the inhibitory effect of CFS derived from probiotics with some food-poisoning bacteria, our recent study successfully declares the inhibitory effect against pathogenic bacteria carrying antimicrobial resistance genes isolated from boar semen. However, further studies are needed to identify the constituent antimicrobial compound in this CFS, construct and synthetic these AMPs. Furthermore, the interaction between synthetic antimicrobial peptides and spermatozoa and the synthetic antimicrobial peptides-based semen extender without antibiotics will be included in a further study to determine its effect on the qualities of spermatozoa and field fertility.

Conclusions
In conclusion, using bacterial contaminated fresh boar semen for artificial insemination is a possible cause of sow endometritis. Consequently, antibiotics are added to the boar semen extender for artificial insemination. Fortunately, CFS derived from probiotics can effectively inhibit bacteria carrying antimicrobial resistance genes isolated from fresh boar semen, in particular the CFS produced from the LAB. However, the ability against bacteria is observed and decreased with more extended incubation periods. In the present results, CFS with its antimicrobial peptides and/or bacteriocin constituent inhibits semen-origin pathogenic bacteria provide the direction as a promising alternative antibiotics method used in the semen extender preservation of the pig industry.

Materials and methods
Sample collection. Total semen samples (n = 10) were collected from 10 individual boars in three distinct AI centers in commercial pig farms in Thailand. Boars were housed in an individual pen in an evaporative cooling house system. They were fed with a commercial feed 3 kg once a day and the water were ad libitum. The sampling locations were conducted in Chai Nat province (farm A; n = 5), Chon Buri province (farm B; n = 3), and Chachoengsao province (farm C; n = 2). The boar semen samples were collected using the gloved hand technique and the whole ejaculate was filtrated with sterile gauze to eliminate the gel-rich fraction 7 . Then, only a fresh sperm-rich fraction was stored in sterile container. All specimens were preserved under the sterile reposi- Bacterial isolation and species identification. All semen samples were cultured on tryptone soy agar (Oxoid, UK) with 5% sheep blood and MacConkey agar (Oxoid, UK) incubated at 37 °C for 18-24 h. All different colonies were identified using standard biochemical tests followed by 16S rRNA sequencing and stored in Brain Heart Infusion (BHI) (Oxoid, UK) with 20% glycerol at -80 °C. Genomic DNA of all isolates was performed using G-spin TM genomic DNA extraction kit (iNtRON, Republic of Korea) and amplified 16S rRNA by PCR with a BiometraTOne96G thermal cycler (AnalytikJena, Germany) using UFUL (5'-GCC TAA CAC ATG CAA GTC GA-3') and 800R (5'-TAC CAG GGT ATC TAA TCC -3') primers. The PCR was performed with the following protocol: initial denaturation at 94 °C for 3 min followed by 30 cycles of denaturation at 94 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 °C for 45 sec, with a final extension step at 72 °C for 5 min. The PCR products were purified by MEGAquick-spin TM Plus Total Fragment DNA purification kit (iNtRON, Republic of Korea) and sequenced with an Applied Biosystems 3730XL DNA Analyzer (Bionics, Republic of Korea). Each 16S rRNA sequences was blasted against the NCBI nucleotide database (https:// blast. ncbi. nlm. nih. gov) to identify all isolates.
Antimicrobial susceptibility testing (AST). All isolates were streaked onto blood agar to obtain single colony. Following incubation at 37 °C for 18-24 h, one to three colonies with similar morphological appearance were transferred into normal saline solution (0.85% NaCl) and thoroughly mixed. The turbidity of bacterial suspension was measured using 0.5 McFarland standard (approximately 10 8 CFU/mL). The minimum inhibitory concentrations (MIC) were conducted by the broth microdilution with following a guideline from the Clinical and Laboratory Standards Institute (CLSI). The assays were performed in triplicate with 96 well plates; in each well, 100 μL of bacterial suspension previously diluted in Mueller Hinton broth (Difco, USA) to 10 6 CFU/mL were added to 100 μL of appropriate dilutions of antimicrobials. A total of 10 antimicrobials were tested in the following concentrations by means of two-fold dilution: amoxicillin (TCI, Japan) 1-128 μg/mL, amoxicillin trihydrate:potassium clavulanate (4:1; Sigma, Germany) 1-128 μg/mL, ceftazidime (Sigma, Germany) 1-128 μg/ mL, ceftriaxone (TCI, Japan) 0.25-32 μg/mL, ceftiofur (TCI,Japan) 0.25-32 μg/mL, colistin (Sigma, Germany) 0.25-32 μg/mL, enrofloxacin (Fluka Biochemika, Japan) 0.06-8 μg/mL, gentamicin (TCI, Japan) 0.5-64 μg/mL, oxytetracycline (AppliChem, USA) 0.25-32 μg/mL, and trimethoprim:sulfamethoxazole (1:19; TCI, Japan) 0.25-32 μg/mL. The 96 well plates were incubated at 37 °C for 16-20 h. Medium without antimicrobials was conducted as control and inoculated prior to and following each antimicrobial-containing series of plates. MIC values were recorded after incubation and defined as the lowest concentration of each antibiotic without a visible growth Agar well diffusion assay. Pseudomonas aeruginosa, E. coli, and Proteus mirabilis isolated from boar semen were subjected for testing the inhibitory effect of CFS from probiotics. All bacteria were cultured in BHI broth at 37 °C for 20-24 h. Bacterial suspension was initially diluted into to 0.5 McFarland standard and performed spread plate method onto nutrient agar. Then, the inoculated nutrient agar was pierced with the sterile 8 mm diameter cork borer to create wells. The volume of 100 μL of CFS was loaded into wells and incubated at 37 °C for 8, 10, 12, 14, and 16 h. After incubation, the inhibition zone will be measured in each well. To validate the result, MRS broth (pH 6.0) was conducted as a negative control.
Statistical analysis. The descriptive statistic was used in this study. In addition, the data analysis was performed by using one-way analysis of variance (ANOVA) and compared means by using Duncan's test by The PASW Statistics for Windows, version 18.0 (SPSS Inc., Chicago, IL, USA). A statistical significance is determined as p value < 0.05.